New indication of cinacalcet hcl pharmaceutical composition for treating cancer

ABSTRACT

A method for treating a cancer includes administering to a subject in need thereof a pharmaceutical composition containing a therapeutically effective amount of Cinacalcet HCl or a pharmaceutical acceptable salt thereof. The cancer is selected from the group consisting of a pleural-related cancer, an abdominal-related cancer, an endocrine-related cancer, a gastrointestinal tract-related cancer, osteosarcoma, skin cancer, and blood cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a National Phase Application filed under 35 U.S.C. 371 as a national stage of PCT/CN2015/092776 filed Oct. 23, 2015, an application claiming the benefit under 35 USC 119(e) to the following U.S. Provisional Applications No. 62/068,298 filed Oct. 24, 2014, the content of each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention related to a new indication of Cinacalcet hydrochloride (Cinacalcet HCl) pharmaceutical composition, especially related to inhibition effect of Cinacalcet hydrochloride (Cinacalcet HCl) pharmaceutical composition on a variety of cancer cells.

BACKGROUND OF THE INVENTION

Cancer is the most popular disease cause of death in the world. The cancer patients are gradually increase yearly, therefore the treatment method of the cancer has become an important issue. The medical treatments of cancer can be classified as surgical treatment, radiation therapy, chemotherapy and target therapy.

Generally, the cancer drug, whether chemotherapy drug or target therapy drug, is to inhibit cancer cells duplication and split to prevent the tumor growth and metastasis.

More attention of therapies is being focused on drugs or other substances that block the growth and spread of cancer by interfering with specific molecules (“molecular targets”) that are involved in the growth, progression, and spread of cancer. Averagely, only about five of 10,000 new drugs can successfully enter the phase I of clinical trials.

Otherwise, the manufacturing of the drug is still a big problem. When the drug starting the clinical trials, there are lots of problems need to overcome, such as drug safety, patient selection, trial dose and other issues. Even the drug has approved by the FDA and sales on the market, there still possibly face the situation of the poor drug response in patients. Furthermore, if the cancer patients happen the drug resistance, that would reduce the effectiveness of the drugs and result in the medical treatment failure. Therefore, the new drug development is very difficult.

Cinacalcet hydrochloride (Cinacalcet HCl) or calcimimetic is a naphthalene derivative and calcimimetic agent that increases the sensitivity of parathyroid gland calcium-sensing receptors to serum calcium. (Cinacalcet HCl) is the orally bioavailable hydrochloride salt of the calcimimetic cinacalcet. Cinacalcet increases the sensitivity of calcium-sensing receptors on chief cells in the parathyroid gland to extracellular calcium, thereby reducing parathyroid hormone (PTH) secretion. In other word, these actions could reduce parathyroid hormone secretion and decreases serum calcium in the treatment of parathyroid disease. Further, a reduction in PTH levels inhibits osteoclast activity, which may result in a decrease in cortical bone turnover and bone fibrosis, and normalization of serum calcium and phosphorus levels. In another way, Cinacalcet HCl therapy was used for secondary hyperparathyroidism in hemodialysis patients. As a result, Cinacalcet HCl is approved by FDA and accumulated a huge data of drug use and drug mechanism research.

Due to the differences of the clinical use, there is no research present that the Cinacalcet HCl has any potential to inhibit cancer cell.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention provides the development of new cancer clinical indications of Cinacalcet HCl.

Accordingly, the present invention provides a new indication of Cinacalcet HCl. The experimental results showed that the Cinacalcet HCl had no toxicity or had little toxicity to normal cells in the present invention. However, the selective effect of Cinacalcet HCl between normal cells and cancer cells need to be identified.

The present invention provides a pharmaceutical composition of Cinacalcet HCl for treating cancer. The pharmaceutical composition is composed of effective dose of Cinacalcet HCl and a pharmaceutical acceptable salt.

In one embodiment of the present invention, the cancer is selected from pleural-related cancer, abdominal-related cancer, endocrine-related cancer, gastrointestinal tract-related cancer.

In one embodiment of the present invention, the cancer is selected from osteosarcoma, skin cancer and blood cancer.

In one embodiment of the present invention, the pleural-related cancer is lung cancer.

In one embodiment of the present invention, the abdominal-related cancer is selected from bladder cancer, and cervical cancer.

In one embodiment of the present invention, the endocrine-related cancer is selected from prostate cancer, breast cancer, and ovarian cancer.

In one embodiment of the present invention, the gastrointestinal tract-related cancer is selected from gastric cancer, hepatic cancer, colorectal cancer, pancreatic cancer, and tongue cancer.

In one embodiment of the present invention, the effective dose of Cinacalcet HCl is from 10 mg/kg/day to 500 mg/kg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG 1 shows the inhibitory effect of Cinacalcet HCl on the different cancer cells.

FIG. 2 shows the results of the inhibitory effect of tumor volume by Cinacalcet HCl.

FIG. 3 shows the inhibitory effect of tumor growth via administered high-dose and low-dose of Cinacalcet HCl.

DETAILED DESCRIPTION OF THE INVENTION

Cell Culture

Subculture the different types of cancer cells. The cancer cells includes lung cancer, gastric cancer, hepatic cancer, colon cancer, skin cancer, cervical cancer, prostate cancer, bladder cancer, breast cancer, leukemia, pancreatic cancer, ovarian cancer, tongue cancer, osteosarcoma, and renal cancer. The normal cells used in the control group included kidney cell (HEK293) and human bronchial epithelial cell line BEAS-2B (as shown in Table 1).

Cancer cell lines were cultured in different culture medium according to different characteristics (as shown in Table 1). The cell numbers were counted and reseed as 2×10⁶ in culture plate/flask. Then, the culture medium were added to a volume of 10 ml, and the cells were cultured for 2-3 days. Then, the cells were suspended for loading into 96-well plates. The number of cells was 3000 and the volume of the culture medium was 100 μl each well.

TABLE 1 Cancer cell lines and culture medium No Cancer type Cancer cell type Culture medium 1 lung cancer H1650 (lung adenocarcinoma) RPMI-1640 A549 (lung adenocarcinoma) DMEM 2 gastric cancer AGS (Gastric Adenocarcinoma) RPMI-1640 MKN-45 (Gastric Adenocarcinoma) RPMI-1640 3 hepatic cancer HepG2 (hepatocellular carcinoma) DMEM Hep3B (hepatocellular carcinoma) DMEM 4 colon cancer HCT116(p53+) (colorectal carcinoma) DMEM LoVo(Colorectal Adenocarcinoma) DMEM 5 skin cancer A375 (amelanotic melanoma) DMEM BCC (basal cell carcinoma) DMEM 6 cervical cancer HeLa (Cervix Adenocarcinoma) DMEM C-33A (Cervical carcinoma) BCRC60554 MEM 7 prostate cancer PC3 (p53−)(Prostate adenocarcinoma) DMEM LNCaP clone FGC (LNCap.FGC) RPMI-1640 8 bladder cancer 8301 (urinary bladder carcinoma) RPMI-1640 T24 RPMI-1640 9 breast cancer MCF7 (Mammary Gland, Adenocarcinoma) DMEM MDA-MB-231 (Mammary Gland, Adenocarcinoma) DMEM 10 pancreatic cancer BxPC-3 RPMI-1640 AsPC-1 RPMI-1640 11 ovarian cancer NIH: OVCAR-3 RPMI-1640 TOV-21G RPMI-1640 12 tongue cancer SAS (Tongue squamous cell carcinoma) DMEM 13 osteosarcoma U-2OS DMEM 14 renal cancer 786-O (Renal adenocarcinoma) BCRC 60243 RPMI-1640 15 normal cell kidney HEK293 (Kidney) DMEM pulmonary epithelial BEAS-2B (Lung Epithelial) RPMI-1640 cell line

Cell Viability Analysis

Removing the original culture medium from 96-well plate. Then add 100 μl of commercially drug at a concentration of 10 μM per well. After 72 hours, add the diluted WST-1 reagent to the well with 100 μl/well, and the diluted WST-1 reagent was acquired from the dilution of 9:1 medium and WST-1 stock reagent. Finally, the total volume of each well was 200 μl/well. Culture the 96-well plate at 37° C. for 30 to 90 minutes. Detecting and calculate the survival rate of each cancer cells with an ELISA reader at OD450 nm. The lower viability of cancer cells represents better inhibition effect via the Cinacalcet HCl drug. Otherwise, the higher viability of cancer cells represents worse inhibition effect via the Cinacalcet HCl drug.

The Effect of Cinacalcet HCl on Different Cancer Cell Lines

The Inhibition Effect of Cinacalcet HCl on Pleural-Related Cancer Cells

This inhibition test of Cinacalcet HCl on pleural-related cancer cells were using two lung cancer cell lines A549 and H1650. The inhibitory tests of Cinacalcet HCl were performed 4 times for each cell lines and then the average value of the inhibitory tests was calculated. The results were shown in Table 2.

TABLE 2 The inhibition effect of Cinacalcet HCl on pleural-related cancer cells 0524-10 min 0526-10 min 0529-10 min 0531-10 min Average A549 87.7 102.7 75.5 92.6 89.6 1-10 min 2-20 min 3-20 min 4-20 min Average H1650 86.8 67.5 76.8 75.2 76.5

The Inhibition Effect of Cinacalcet HCl on Abdominal-Related Cancer Cell Lines

This inhibition test of Cinacalcet HCl on abdominal-related cancer cells were using bladder cancer cell line TSGH and T24 (Table 3), cervical cancer cell lines HeLa and C-33A (Table 4), renal cancer cell line 786-O (Table 5). The inhibitory tests of Cinacalcet HCl were performed 4 times for each cell lines and then the average value of the inhibitory tests was calculated. The results were shown in Table 3, Table 4, and Table 5.

TABLE 3 The inhibition effect of Cinacalcet HCl on bladder cancer cell lines 0510-10 min 0512-10 min 0515-10 min 0517-10 min average TSGH 57.3 73.2 74.7 33.9 59.8 T24-1-30 T24-2-20 T24-3-20 T24-4-20 min min min min average T24 71.1 132.6 177.1 103.3 121.0

TABLE 4 The inhibition effect of Cinacalcet HCl on cervical cancer cell lines 0524-10 0526-10 0529-10 0531-10 min min min min average HeLa 109.6 118.6 104.1 113.0 111.3 C-33A 114.5 118.8 108.1 100.0 110.3

TABLE 5 The inhibition effect of Cinacalcet HCl on renal cancer cell lines 0524-10 min 0526-10 min 0529-10 min 0531-10 min average 786-O 81.3 71.6 80.2 87.1 80.0

The Inhibition Effect of Cinacalcet HCl on Endocrine-Related Cancer Cell Lines

This inhibition test of Cinacalcet HCl on endocrine-related cancer cells were using prostate cancer cell lines PC-3 and LNCap (Table 6), breast cancer cell lines MCF7 and MDA-MB-231 (Table 7), and ovarian cancer cell lines NIH-OVCAR-3 and TOV-21G (Table 8). The inhibitory tests of Cinacalcet HCl were performed 4 times for each cell lines and then the average value of the inhibitory tests was calculated. The results were shown in Table 6, Table 7, and Table 8.

TABLE 6 The inhibition effect of Cinacalcet HCl on prostate cancer cell lines PC-3- PC-3- PC-3- PC-3- 0524-10 0526-10 0529-10 0531-10 min min min min average PC-3 102.7 132.2 120.3 134.6 122.5 Average LNCap 52.6

TABLE 7 The inhibition effect of Cinacalcet HCl on breast cancer cell lines 0612-10 0614-10 0616-10 0619-10 min min min min average MCF7 87.7  75.6  82.2  77.2  80.7  MDA- 92.35 84.94 60.81 92.75 82.71 MB-231

TABLE 8 The inhibition effect of Cinacalcet HCl on ovarian cancer cell lines 7-3-30 min 7-4-30 min 7-7-30 min -4-30 min average NIH-OVCAR-3  90.7 111.1 100.2 111.0 103.3 TOV-21G 128.7 109.4 104.1 108.2 112.6

The Inhibition Effect of Cinacalcet HCl on Gastrointestinal Tract-Related Cancer Cell Lines

This inhibition test of Cinacalcet HCl on gastrointestinal tract-related cancer cells were using gastric cancer cell lines AGS and MKN-45 (Table 9), hepatic cancer cell lines HepG2 and Hep3B (Table 10), colorectal cancer cell lines HCT116-wt and LoVo (Table 11), pancreatic cancer cell line AsPC-1 and BxPC-3(Table 12), tongue cancer cell line SAS (Table 13). The inhibitory tests of Cinacalcet HCl were performed 4 times for each cell lines and then the average value of the inhibitory tests was calculated. The results were shown in Table 9, Table 10, Table 11, Table 12 and Table 13.

TABLE 9 The inhibition effect of Cinacalcet HCl on gastric cancer cell lines 0510-10 0512-10 0515-10 0517-10 min min min min average AGS 26.6  20.0  52.3 14.50856943 28.3 MKN-45 82.1 101.5 103.6 69.1     89.1

TABLE 10 The inhibition effect of Cinacalcet HCl on hepatic cancer cell lines 0524-20 0526-20 0529-20 0531-20 min min min min average HepG2 105.7 132.6 116.5  89.5 111.1 0612-20 0614-20 0616-20 0619-20 min min min min average Hep3B 143.2 125.1 151.5 116.8 134.1

TABLE 11 The inhibition effect of Cinacalcet HCl on colorectal cancer cell lines 0602-30 0605-10 0607-10 0609-10 min min min min average HCT116-wt 89.0 107.3 132.5 112.6 110.3 0616-10 0619-10 0621-10 0623-10 min min min min average LoVo 73.9 125.0  90.4  84.2  93.4

TABLE 12 The inhibition effect of Cinacalcet HCl on pancreatic cancer cell lines 1-7-3-30 min 1-7-4-30 min 1-7-7-30 min 1-4-30 min Average AsPC-1 61.6 81.7  60.4 71.9 68.9 3-7-3-30 min 3-7-4-30 min 3-7-7-30 min 3-4-30 min Average BxPC-3 64.6 78.8 100.1 76.2 79.9

TABLE 13 The inhibition effect of Cinacalcet HCl on tongue cancer cell lines 6-26-10 min 6-28-10 min 6-30-10 min 7-3-10 min average SAS 45.7 88.9 96.2 98.9 82.4

The Inhibition Effect of Cinacalcet HCl on Other Cancer Cell Lines

This inhibition test of Cinacalcet HCl on other cancer cells were using osteosarcoma cell line U2OS (Table 14), skin cancer cell lines A375 and BCC (Table 15). The inhibitory tests of Cinacalcet HCl were performed 4 times for each cell lines and then the average value of the inhibitory tests was calculated. The results were shown in Table 14 and Table 15.

TABLE 14 The inhibition effect of Cinacalcet HCl on osteosarcoma cancer cell line 6-26-10 min 6-28-10 min 6-30-10 min 7-3-10 min average U2OS 82.6 86.7 64.9 75.3 77.4

TABLE 15 The inhibition effect of Cinacalcet HCl on skin cancer cell lines 0602-30 min 0605-10 min 0607-10 min 0609-10 min average A375 117.1  95.6  94.4 108.5 103.9 0602-30 min 0605-10 min 0607-10 min average BCC 114.2 105.5 112.6 110.8

The Experiment Design on Control Group

The Inhibition Effect of Cinacalcet HCl on Normal Cells

This inhibition test of Cinacalcet HCl on normal cells were using normal kidney cell line HEK293 (Table 16), normal pulmonary epithelial cell lines canine fibroblast cell line BEAS-2B (Table 17). The inhibitory tests of Cinacalcet HCl were performed 4 times for each cell lines and then the average value of the inhibitory tests was calculated The results were shown in Tables 16 and Tables 17.

TABLE 16 The inhibition effect of Cinacalcet HCl on normal kidney cell line average HEK293 114.1

TABLE 17 The inhibition effect of Cinacalcet HCl on normal pulmonary epithelial cell line 0510-10 0512-10 0515-10 0517-10 min min min min average BEAS-2B 70.5 72.9 72.2 89.0 76.1

This inhibition test results of Cinacalcet HCl on all kinds of cells were shown in Table 18. Cinacalcet HCl is having different inhibitory effect on different tumor cell type even in the same cancer. As a result in the experiments of the present invention, Cinacalcet HCl has a significant inhibitory effect and specificity on various cancer cells. (FIG. 1)

TABLE 18 Summary of the Effect on different cancer cell lines by Cinacalcet HCl cancer cells Inhibitory effect lung cancer 83.09384194 bladder cancer 90.4 cervical cancer 111.32 Kidney cancer 87.53 prostate cancer 81.70 breast cancer 107.9 ovarian cancer 58.7 gastric cancer 122.60 hepatic cancer 101.85 colorectal cancer 74.4 pancreatic cancer 82.44 tongue cancer 77.37 osteosarcoma 107.32 skin cancer 80.0 kidney cell lines 114.06 normal pulmonary epithelial 76.13 cell line

Animal Model Test of Gastric Cancer with Dose 100 mg/kg/day and 200 mg/kg/day

In this invention, the female mice were(BALB/cAnN.Cg-Foxnl™/CrlNarl) and purchased from National Laboratory Animal Center (Taiwan). The weight of the mice were 21±1 g. These mice were subcutaneously injected with gastric cancer cells (AGS) and then put these mice into different cages at random. The drug test experiment was divided into three groups, include “control group”, “low dose group (100 mg/kg/day)”, “high dose group (200 mg/kg/day)”. These mice were then injected test drug intraperitoneally once daily until the tumor size reached 100 mm³. The tumor sizes and body weight were measured twice a week. The tumor sizes were measured and calculated by formula: (L×W²)/2. L represents the tumor longest length. W represents the tumor shortest diameter. The experiment result is shown in Table 19.

TABLE 19 The inhibitory effect of tumor volume via administered Cinacalcet HCl control group Low dose (100 mg/kg/day) high dose (200 mg/kg/day) tumor tumor tumor longest vol- volume longest volume longest volume weight length width ume growth weight length width volume growth weight length width volume growth (g) mm mm mm3 mm3 (g) mm mm mm3 mm3 (g) mm mm mm3 mm3 First measurement A 18.5 7 7 171.5 171.5 20 11 8 352 352 19 7 5 87.5 87.5 B 22 8 6 144 144 16 13 8 416 416 19.5 6 6 108 108 C 20.5 9 8 288 288 21 14 12 1008 1008 1.5 6 4 48 48 aver- 20.4 7.6 7 189.3 189.3 592 592 81.16667 81.16667 age Second measurement A 22 7 6 126 −45.5 21 8 5 100 −252 18.5 7 7 171.5 84 B 20 8 7 196 52 22 16 8 512 96 21 6 7 147 39 C 20 9 7 220.5 −67.5 19 9 7 220.5 −787.5 19 6 5 75 27 aver- 20.6 8.4 6.8 198.5 9.2 277.5 −314.5 131.1667 50 age Third measurement A 23 9 6 162 36 23.5 19 15 2137.5 2037.5 19 7 5 87.5 −84 B 20 10 8 320 124 21 7 6 126 −386 22 6 5 75 −72 C 21 11 7 269.5 49 21 8 6 144 −76.5 18.5 6 5 75 0 aver- 21.2 10 6.8 235.3 36.8 802.5 525 79.16667 −52 age Fourth measurement A 22 12 8 384 114.5 22 10 7 245 −1892.5 23 6 3 27 −60.5 B 22 11 8 352 172 22 14 7 343 217 23 5 4 40 −35 C 23 12 9 486 134 23 13 13 1098.5 954.5 20 0 0 0 −75 aver- 22.4 295.7 62.2 562.1667 −240.3333 22.33333 −56.8333 age

According to the results in FIG. 2, both low dose and high dose of Cinacalcet HCl had significant inhibition effect on tumor cells, and the weight of mice did not show a significant decrease during the experiment. These results indicated that both high and low doses of Cinacalcet HCl could keep the tested mice in healthy status during the treatment without death.

According to the results in FIG. 3, both low dose and high dose of Cinacalcet HCl had effectively slow down the tumor volume growth, and can also reduce the tumor volume. Especially, high doses of Cinacalcet HCl had better effect to inhibit tumor growth.

Although the present invention has been described in terms of specific exemplary embodiments and examples, it will be appreciated that the embodiments disclosed wherein are for illustrative purposes only and various modifications and alterations might be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the following claims. 

1. A method for treating a cancer comprising: administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of Cinacalcet HCl or a pharmaceutical acceptable salt thereof.
 2. The method of claim 1, wherein the cancer is selected from the group consisting of a pleural-related cancer, an abdominal-related cancer, an endocrine-related cancer, and a gastrointestinal tract-related cancer.
 3. The method of claim 1, wherein the cancer is selected from the group consisting of osteosarcoma, skin cancer, and blood cancer.
 4. The method of claim 2, wherein the pleural-related cancer is lung cancer.
 5. The method of claim 2, wherein the abdominal-related cancer is selected from the group consisting of bladder cancer, cervical cancer, and kidney cancer.
 6. The method of claim 2, wherein the endocrine-related cancer is selected from the group consisting of prostate cancer, breast cancer, and ovarian cancer.
 7. The method of claim 2, wherein the gastrointestinal tract-related cancer is selected from the group consisting of gastric cancer, hepatic cancer, colorectal cancer, pancreatic cancer, and tongue cancer.
 8. The method of claim 1, wherein the effective amount of Cinacalcet HCl is from 10 mg/kg/day to 500 mg/kg/day. 